Innate immune responses provide the first line of defense against invading pathogens through the recognition of pathogen associated molecular patterns. The RIG-I like receptors (RLRs), RIG-I, MDA5 and LGP2, are cytosolic sensors of viral RNA and play pivotal roles in innate antiviral immune responses. The RLRs recognize 5' triphosphate single-stranded RNA or double-stranded RNA, signature structures of viral RNA. Stimulation of the RLRs by viral RNA leads to the induction of type I interferons and other cytokines, conferring antiviral activity to the host and activating the acquired immune responses. However, the detailed mechanisms of viral RNA sensing and signaling by the RLRs are still not fully understood. It is not clear what are the structural features of viral RNA recognized by the RLRs and how the RLRs recognize viral RNA at molecular level. The mechanisms of RLRs activation by viral RNA are still largely unknown. The proposed research will focus on elucidating the structural basis of viral RNA sensing by the RLRs with the following specific aims: 1) determine the structural features of RNA recognized by the RLRs; 2) elucidate the structural basis of RNA recognition by the RLRs; 3) investigate the molecular mechanism of RLRs activation by RNA. This research represents a vigorous and comprehensive investigation of the structural basis for viral RNA sensing by the RLRs through a combined approach of biochemical, structural, and functional studies. These studies will provide important insights into the molecular mechanism of viral RNA sensing by the RLRs. This research will significantly advance our understanding about the mechanisms of antiviral immune response mediated by the RLRs and provide a structural framework for the development of more effective adjuvant for vaccines and novel therapeutic reagents to modulate the antiviral immune responses. PUBLIC HEALTH RELEVANCE: In this research, we will elucidate how our immune system responds to viral infections through studying the molecular structures of key proteins sensing the viral nucleic acids. These studies will lead to the fundamental understanding about the molecular mechanism of antiviral immunity and will also provide critical information for potential therapeutic interventions.